Methods and means for detecting enzymatic cleavage and linkage reactions

Abstract

The invention relates to methods and means for the detection of enzyme-catalyzed cleavage and linking reactions. Modular chemical compounds which serve as substrates for the enzymes involved are provided. The detection of the reaction products is effected by means of molecular-weight-sensitive methods.

1 . A method for the detection of enzyme-catalyzed cleavage reactions, comprising the following steps: 
 a) providing a modular chemical compound containing the following sequence motif:    Z-X—Y or Y—X-Z     wherein Z comprises a module with a selectable molecular weight which is inert with respect to said enzymatic cleavage reaction;     X comprises a module with n cleavage sites S 1  to S n  which can be cleaved by said enzyme-catalyzed cleavage reaction into at least two cleavage products containing X 1  and X n+1 , respectively, and having defined molecular weights, n being an integer of ≧1; and     Y comprises a reporter module;     as a substrate for said enzyme(s) catalyzing said cleavage reaction(s);    b) incubating the compound with said enzyme(s) catalyzing said cleavage reaction(s) to form at least two cleavage products which contain    Z-X 1  and X n+1 —Y or Y—X 1  and X n+1 -Z     wherein the molecular weight of the cleavage product containing Z-X 1  or X n+1 -Z is at least fifty percent of the total molecular weight of the substrate;    c) detecting the enzymatic activity or activities by determining the cleavage product containing the reporter module Y by means of a molecular-weight-sensitive method.    
     
     
         2 . The method according to  claim 1 , characterized in that X comprises a module with one cleavage site S 1  which can be cleaved by the enzyme-catalyzed cleavage reaction into two cleavage products containing X 1  and X 2 , respectively, and having defined molecular weights.  
     
     
         3 . The method according to  claim 1 , characterized in that X comprises a module with several cleavage sites S 1  to S n  with n>1 which can be cleaved by enzymatic cleavage reactions into n+1 cleavage products having defined molecular weights.  
     
     
         4 . A method for the detection of enzyme-catalyzed linking reactions, comprising the following steps: 
 a) providing the following chemical compounds:    Z-X 1  and X n+1 —Y and optionally X 2  to X n ; or Y—X 1  and X n+1 -Z and optionally X 2  to X n ;     wherein Z comprises a module with a selectable molecular weight which is inert with respect to said enzymatic linking reaction;     X 1  comprises a modular building block which can be linked by said enzyme-catalyzed linking reaction(s) directly with X n+1  in the case where n=1, or optionally indirectly through at least one modular building block selected from X 2  to X n  in the case where n>1;     X n+1  comprises a modular building block which can be linked by said enzyme-catalyzed linking reaction(s) directly with X 1  in the case where n=1, or optionally indirectly through at least one modular building block selected from X 2  to X n  in the case where n>1;     Y comprises a reporter module;     n is an integer of ≧1; and     the molecular weight of X n+1 —Y or Y—X 1  is at most fifty percent of the total molecular weight of the linked product formed;     as a substrate for said enzyme(s) catalyzing said cleavage reaction;    b) incubating the compound with said enzyme(s) catalyzing said linking reaction(s) to form linked products    Z-X—Y or Y—X-Z     wherein X comprises the modular building blocks X 1  and X n+1  and optionally at least one modular building block selected from X 2  to X n ;    c) detecting the enzymatic activity or activities by determining the linked product containing the reporter module Y by means of a molecular-weight-sensitive method.    
     
     
         5 . The method according to  claim 4 , characterized in that X consists of two modular building blocks X 1  and X 2  which have been directly linked with each other by said enzyme-catalyzed reaction.  
     
     
         6 . The method according to  claim 4 , characterized in that X 1  and X n+1  are indirectly linked with one another through at least one, preferably by a plurality, of modular building blocks selected from X 2  to X n  with enzyme catalysis.  
     
     
         7 . The method according to any of  claims 1  to  3 , characterized in that the molecular weight of Z-X 1  and X n+1 -Z in the cleavage assay is at least about sixty percent, especially at least about seventy percent, preferably at least about eighty percent, more preferably at least about ninety percent, of the molecular weight of compound Z-X—Y or Y—X-Z.  
     
     
         8 . The method according to any of  claims 4  to  6 , characterized in that the molecular weight of Y—X 1  and X n+1 —Y in the linking assay is at most about forty percent, especially at most about thirty percent, preferably at most about twenty percent, more preferably at most about ten percent, of the molecular weight of compound Z-X—Y or Y—X-Z.  
     
     
         9 . The method according to any of  claims 1  to  8 , characterized in that Z comprises a polymer constituted of monomer building blocks.  
     
     
         10 . The method according to any of  claims 1  to  9 , characterized in that the components of compound Z-X—Y or Y—X-Z are or become covalently linked with each other, and/or the components of the individual substrates of the enzymatic linking reaction(s) are covalently linked with each other.  
     
     
         11 . The method according to any of  claims 1  to  10 , characterized in that X and Z belong to different classes of substances.  
     
     
         12 . The method according to any of  claims 1  to  11 , characterized in that X and/or Z belong to the following classes of substances: synthetic polymers, dendrimers, natural substances, nucleic acids, peptide nucleic acids (PNA), peptides, proteins, lipids, carbohydrates or derivatives of the above mentioned substances.  
     
     
         13 . The method according to any of  claims 1  to  12 , characterized in that Z comprises a double-stranded nucleic acid or a double-stranded nucleic acid derivative.  
     
     
         14 . The method according to any of  claims 1  to  3  and  7  to  13 , characterized in that an enzyme catalyzing the reaction is a hydrolase, especially a lipase, phosphatase, amylase, glycosidase, nuclease, peptidase, protease, amidase, pyrophosphatase, ATPase, sulfatase or phosphoamidase.  
     
     
         15 . The method according to any of  claims 1  to  3  and  7  to  14 , characterized in that said enzyme catalyzing the reaction is a lyase, especially a C—C, C—O, C—N or C—S lyase.  
     
     
         16 . The method according to  claim 15 , characterized in that said lyase is a (de)carboxylase, aldolase, dehydratase, ammonia-lyase, arginosuccinase or cysteine desulfhydrase.  
     
     
         17 . The method according to any of  claims 4  to  13 , characterized in that said enzyme catalyzing the reaction is a ligase, especially a C—O, C—S, C—N or C—C ligase.  
     
     
         18 . The method according to  claim 17 , characterized in that said ligase is an amino-acid activating ligase, acyl-CoA synthetase, glutamine synthetase or pyruvate carboxylase.  
     
     
         19 . The method according to any of  claims 4  to  13 , characterized in that said enzyme catalyzing the reaction is a transferase, especially a DNA or RNA polymerase.  
     
     
         20 . The method according to any of  claims 1  to  19 , characterized in that said enzyme-catalyzed cleavage or linking reactions are performed in the presence of cosubstrates and/or cofactors.  
     
     
         21 . The method according to any of  claims 1  to  20 , characterized in that said reporter module Y is luminescent, especially fluorescent.  
     
     
         22 . The method according to any of  claims 1  to  21 , characterized in that the detection method determines a measure of the rotational or translational diffusion rate of the cleavage or linked product containing said reporter module Y.  
     
     
         23 . The method according to claims  21  or  22 , characterized in that the detection method is based on fluorescence spectroscopy, especially confocal fluorescence spectroscopy.  
     
     
         24 . The method according to any of  claims 21  to  23 , characterized in that the detection method is based on fluorescence correlation spectroscopy (FCS), fluorescence polarization determination and/or fluorescence anisotropy determination.  
     
     
         25 . The method according to any of  claims 1  to  24 , characterized in that said method is performed as a homogeneous assay.  
     
     
         26 . The method according to any of  claims 1  to  25 , characterized in that one or more enzymes and/or substrates are employed simultaneously or sequentially.  
     
     
         27 . The method according to any of  claims 1  to  26 , characterized in that the enzyme or enzymes catalyzing the cleavage or linking reactions is detached from the reaction product containing the reporter module Y, preferably (bio)chemically, before the enzymatic activity is established by a molecular-weight-sensitive method.  
     
     
         28 . The method according to any of  claims 1  to  27 , characterized by being performed in the presence of modulators or potential modulators of enzymatic activity.  
     
     
         29 . The method according to any of  claims 1  to  28 , characterized by being employed for testing enzyme and/or modulator specificities, for testing enzyme and/or modulator activities, for the identification of modulators and/or substrates, for the screening for pharmacologically active substances, for diagnostic purposes, or for the detection of contaminations in chemical or biological samples.  
     
     
         30 . The method according to any of  claims 1  to  29 , characterized in that enzymes having substrate, group or steric specificity are employed.  
     
     
         31 . The method according to any of  claims 1  to  30 , characterized in that Z has such a chemical structure that Z does not sterically interact with the cleavage or linking site.  
     
     
         32 . A generic modular substrate, especially for performing enzyme-catalyzed cleavage reactions according to any of  claims 1  to  3  and  7  to  31 , containing the following sequence motif:  
       Z-X—Y or Y—X-Z  
       wherein X comprises a module with n cleavage sites S 1  to S n  which can be cleaved by at least one enzyme-catalyzed cleavage reaction into at least two cleavage products containing X 1  and X n+1 , respectively, and having defined molecular weights, n being an integer of ≧1; 
 Y comprises a reporter module; and  
 Z comprises a module with a selectable molecular weight which is inert with respect to said enzymatic cleavage reaction(s) and whose molecular weight has been selected such that the molecular weight of the cleavage product containing Z-X 1  or X n+1 -Z after the enzymatic reaction has occurred is at least fifty percent of the total molecular weight of the generic substrate.  
 
     
     
         33 . The substrate according to  claim 32 , characterized in that the molecular weight of Z is selected in such a way that, after the enzymatic reaction has occurred, the molecular weight of the cleavage product containing Z-X 1  and X n+1 -Z is at least about sixty percent, especially at least about seventy percent, preferably at least about eighty percent, more preferably at least about ninety percent, of the molecular weight of compound Z-X—Y or Y—X-Z.  
     
     
         34 . The substrate according to  claim 32  or  33 , characterized in that X comprises a module with one cleavage site Si which can be cleaved by the enzyme-catalyzed cleavage reaction into two cleavage products containing X 1  and X 2 , respectively, and having defined molecular weights.  
     
     
         35 . The substrate according to  claim 32  or  33 , characterized in that X comprises a module with several cleavage sites S 1  to S n  with n>1 which can be cleaved by enzymatic cleavage reactions into n+1 cleavage products having defined molecular weights.  
     
     
         36 . A kit containing a substrate according to any of  claims 32  to  35  and at least one enzyme for catalyzing said cleavage reaction(s) at the cleavage sites S 1  to S n , and optionally cosubstrates, cofactors, modulators, buffers and/or reagents for stopping said enzymatic reaction.  
     
     
         37 . Use of a substrate according to any of  claims 32  to  35  or of a kit according to  claim 36  in enzymatic cleavage reactions.  
     
     
         38 . A kit, especially for performing enzyme-catalyzed linking reactions according to any of  claims 4  to  31 , comprising the following chemical compounds as substrates for said enzyme or enzymes catalyzing the linking reaction(s):  
       Z-X 1  and X n+1 —Y and optionally X 2  to X n ; or Y—X 1  and X n+1 -Z and optionally X 2  to X n ;  
       wherein 
 X 1  comprises a modular building block which can be linked by said enzyme-catalyzed linking reaction(s) directly with X n+1  in the case where n=1, or optionally indirectly through at least one modular building block selected from X 2  to X n  in the case where n>1;  
 X n+1  comprises a modular building block which can be linked by said enzyme-catalyzed linking reaction(s) directly with X 1  in the case where n=1, or optionally indirectly through at least one modular building block selected from X 2  to X n  in the case where n>1;  
 Y comprises a reporter module;  
 n is an integer of ≧1; and  
 Z comprises a module with a selectable molecular weight which is inert with respect to said enzymatic linking reaction and whose molecular weight has been selected such that the molecular weight of X n+1 —Y or Y—X 1  is at most fifty percent of the total molecular weight of the linked product formed;  
 and optionally at least one enzyme catalyzing said linking reaction, cosubstrates, cofactors, modulators, buffers and/or reagents for stopping said enzymatic reaction.  
 
     
     
         39 . A kit according to  claim 38 , characterized in that the molecular weight of Z is selected in such a way that the molecular weight of Y—X 1  and X n+1 —Y is at most about forty percent, especially at most about thirty percent, preferably at most about twenty percent, more preferably about ten percent, of the molecular weight of the compound Z-X—Y or Y—X-Z.  
     
     
         40 . Use of a kit according to  claim 38  or  39  in enzymatic linking reactions.  
     
     
         41 . Use of a substrate according to any of  claims 32  to  35  or of a kit according to any of  claims 36  to  39  for testing enzyme and/or modulator specificities, for testing enzyme and/or modulator activities, for the identification of modulators and/or substrates, for the detection of contaminations in chemical or biological samples, for the screening for pharmacologically active substances, or for diagnostic purposes.  
     
     
         42 . A method for the detection of contaminations in at least one reagent which is to be used for performing an enzymatic cleavage reaction in which either a modular chemical compound Z-X—Y is cleaved into at least two cleavage products Z-X 1  and X n+1 —Y, or a modular chemical compound Z-X—Y is cleaved into at least two cleavage products Y—X 1  and X n+1 -Z, comprising the following steps: 
 a) providing a modular chemical compound containing the following sequence motif:  
 Z-X—Y or Y—X-Z  
  wherein Z comprises a module with a selectable molecular weight which is inert with respect to said enzymatic cleavage reaction and whose molecular weight has been selected such that the molecular weight of the cleavage product containing Z-X 1  or X n+1 -Z after the chemical reaction has occurred is at least fifty percent of the total molecular weight of said modular chemical compound;  
  X comprises a module with n cleavage sites S 1  to S n  which can be cleaved by said enzyme-catalyzed cleavage reaction into at least two cleavage products containing X 1  and X n+1 , respectively, and having defined molecular weights, n being an integer of ≧1; and  
  Y comprises a reporter module;  
 b) incubating said compound with said reagent;  
 c) detecting contaminations by determining a compound containing the reporter module Y by means of a molecular-weight-sensitive method.  
 
     
     
         43 . The method according to  claim 42 , characterized in that said reagent comprises the enzyme catalyzing the cleavage reaction as well as an inhibitor specific for this enzyme.